The study and characterization of proteins and peptides has become such a significant part of modern biology that it has its own name: Proteomics. Mass spectrometry has become one of the most important techniques used for the analysis of peptides and proteins, and a number of different mass spectrometry experiments are performed in this field. The present invention relates to the use of mass spectrometry to characterize the sequence of amino acids in peptides and proteins, using either the “bottom up” or the “top down” techniques as have been previously described in the literature. Presently, the most widely used of these types of experiments is the “bottom up” proteomics experiment. However, the present invention described herein will significantly advance the practice of the “top down”-type experiments, as well as any proteomic mass spectrometry experiment which utilizes tandem mass spectrometry (MS/MS).
In the “bottom up”-type experiment, mixtures of proteins, usually derived from some biological sample (such as a cell lysate) and therefore potentially containing as many as several thousand proteins with relative abundances ranging over several orders of magnitude, are analyzed. Such protein samples are digested with a proteolytic enzyme (typically trypsin, or a combination of trypsin and endo-Lys) resulting in a complex mixture of tryptic peptides (the digestion typically yields about 30 peptides/protein). After the digestion step, there generally are various steps of sample cleanup, separation, fractionation and/or chemical derivatization prior to the introduction of the sample to the mass spectrometer. In one embodiment, the processed peptide samples are chromatographically separated and introduced to the mass spectrometer by means of a nanoflow-HPLC (5-200 nL/min) interfaced directly to an electrospray ionization source on one of three different types of mass spectrometers: Finnigan LCQ Deca or LCQ XP (RF 3D quadruole ion traps), Finnigan LTQ (radial ejection RF 2D quadrupole ion trap) or Finnigan LTQ/FT instruments (tandem RF 2D quadrupole ion trap/Fourier transform ion cyclotron resonance mass spectrometer).
The electrospray ionization source converts neutral sample peptides, eluting from the HPLC column, to ions in the gas-phase for analysis by the mass spectrometer. In an aqueous acidic solution, tryptic peptides are protonated on both the amino terminus and the side chain of the C-terminal amino acid (Lys or Arg). As the electrosprayed-peptides enter the mass spectrometer, the water is pumped away and the positively charged amino groups both hydrogen bond and transfer protons to the amide groups along the backbone of the peptide. The result is that the aggregate of each tryptic peptide species eluting from the HPLC is converted into a collection of ionized peptide molecules protonated at different sites along the peptide backbone.
In accordance with one procedure, MS/MS spectra of the ions produced from different peptide species are obtained (mass spectra of fragment ions) in the following sequence of steps.
1. Peptide Ions are introduced and trapped in a RF quadrupole ion trap (2D or 3D)
2. All ions outside of a narrow range of mass-to-charge ratios (m/z) associated with the chosen peptide precursor ion species are eliminated from the trap.
3. The isolated precursor peptide ions are kinetically excited and undergo collisionally activated decomposition (CAD).
4. Retained product ions are mass analyzed to produce a mass (m/z) spectrum.
During step 2, the protonated peptides ions undergo several hundred or thousand collisions with helium atoms, which are present at a pressure of about 1-5 millitorr. During this process the internal energy of the ions is increased by small increments until it exceeds the activation energy required to break the protonated amide bond in the backbone of the molecule (this process is also often referred to as collision induced dissociation, CID). Ideally, the result is a collection of b and y-type fragment ions that differ in mass by a single amino acid. FIG. 1 displays the nomenclature describing the various types of peptide backbone cleavage. Type b ions contain the amino terminus plus one or more amino acid residues. Type y ions contain the carboxyl terminus plus one or more amino acid residues. Subtraction of m/z values for fragments of the same type that differ by a single amino acid yield the mass, and thus the identity of the extra residue in the larger of the two fragments. By continuing this process, it is possible to read the amino acid sequence of the targeted peptide backwards (y ions) and forwards (b ions). A skilled analyst can ascertain all or part of the amino acid sequence of the precursor peptide. There are also computer programs that compare peptide MS/MS spectra to theoretical MS/MS spectra of peptides derived from protein and nucleic acid databases to produce a list of likely precursor peptides (and their structures) for each MS/MS spectrum.
The typical sample is quite complex, so tens or hundreds of different peptides may simultaneously elute from the LC column. To give the instrument time to record MS/MS spectra of a larger percentage of the coeluting peptide precursors a procedure referred to as Peak Parking can be used to extend the chromatography to provide sample peak widths from 10 sec to 200 sec. The use of Peak Parking has been previously described in the literature and is known to those skilled in the art. Generally the experiment is automated and involves the repetition of a sequence mass spectral experiments involving a first full scan MS experiment from which precursor m/z peaks are selected (through automated analysis of the resulting m/z spectrum) for subsequent MS/MS analysis. Depending, upon the instrument speed and the chromatographic peak width (duration of elution of individual species), typically anywhere from 3 to 10 MS/MS spectra are acquired for precursor m/z values determined from the initial MS only experiment. The critera for data dependent selection of precursor m/z values are designed so as to minimize the recording of redundant MS/MS spectra and MS/MS spectra of known background and contaminant peaks. A single such data dependent mode LC MS/MS experiment may vary in duration from 30 minutes to 4 hours and thousands of MS/MS spectra of peptide ions may be recorded. At the end of the chromatographic run, proteins in the original mixture are identified by processing the set of recorded MS/MS spectra of peptide precursors against the various protein and nucleic acid databases. Computer programs for analyzing the data are commercially available and include the SEQUEST (marketed by Thermo Electron) and MASCOT (Matix Science) computer programs. Routinely, six thousand sequences of tryptic peptides can be obtained in a single 4 hr chromatographic run with the above technology. Peptides present at the 5-10 fmol level in complex mixtures (loaded on column) are readily identified.
For the analysis of these complex mixtures of peptides, the greater number of unique MS/MS spectra recorded, the more complete the characterization of the mixture and the greater portion of the peptides from source proteins will be observed (sequence coverage), yielding greater certainty in their identification. Hence, the time it takes to obtain a single MS/MS spectrum is important. A mass spectrometer or method of performing MS/MS that isn't capable of producing a MS/MS spectrum in less than about 2 seconds is considered unsuitable for chromatographic applications such as the “bottom up” type proteomics experiment.
The use of CAD for the production of product ions suffers from several disadvantages include the following:
a) Peptides with post-translational modifications (i.e., phosphorylation and glycosylation, etc) often fragment by loss of the modification rather by cleavage of the peptide backbone. Only a relatively small percentage about (20%-30%) of these types of peptide ion precursors produce interpretable/searchable product ion spectra.
b) Peptides that contain multiple basic amino acid residues (Lys, Arg, and His) and thus carry more than two charges, also fail to fragment randomly along the peptide backbone and thus afford incomplete sequence information when analyzed by the above technology.
c) Peptides that contain more than 40 amino acids also fail to fragment randomly along the peptide backbone. These also afford incomplete sequence information.
Accordingly, there is a need for an improved method of fragmenting peptides to produce a suitable array of interpretable/searchable product ion spectra. An alternate strategy for fragmenting protonated peptides and proteins in the gas phase was suggested by McLafferty, et al., in 1998 (J. Am. Chem. Soc. 1998, 120, 3265-3266). This technique involves interacting the protonated peptides with thermal electrons while both are stored inside an ICR cell of a Fourier transform mass spectrometer. This process is referred to as electron capture dissociation (ECD). The originally proposed mechanism for this dissocation process is as follows:
Reaction of a protonated amine group, RNH3+, on a multiply charged peptide with a thermal electron is exothermic by about 6 eV and forms a neutral hypervalent nitrogen species, RNH3 (see FIGS. 2A-C). This compound then dissociates to RNH2 and a hydrogen radical, H., on a time scale that is short compared to energy delocalization via vibrational modes of the molecule. The hydrogen radical attaches to the peptide backbone and triggers cleavage reactions to produce a homologous series of fragment ions of type a, c, y, and z. The c and z type ions are generally more abundant. Again subtraction of m/z values for fragments within a given ion series that differ by a single amino acid affords the mass, and thus the identity of the extra residue in the larger of the two fragments. By continuing this process, it is possible to read the amino acid sequence of the targeted peptide backwards (y and z ions) and forwards (a and c ions). Since this mobile hydrogen radical mechanism was first proposed, alternative mechanisms have been proposed which account for various perceived inadequacies the proposed mechanism such as the capacity of ECD to equivalently fragment multiply sodiated protein and peptide ions (ionized by the addition of Na+ rather that H+).
The advantages of this approach include the following:
1) Peptides with post-translational modifications (phosphorylation or glycosylation) primarily fragment at the peptide backbone bonds and are easily sequenced by mass spectrometry. Fragmentation with loss of the post-translational modification and loss of other side chain moieties is only a minor side reaction or not observed at all.
2) Peptides that contain multiple basic residues (and thus carry more than two positive charges in the gas phase), still fragment more or less randomly along the peptide backbone and are easily sequenced.
3) ECD fragmentation is not limited by the size of the peptide being analyzed. The McLafferty group has now provided extensive evidence that ECD can be employed to confirm the sequences of intact proteins and to locate post-translational modification on the intact molecules.
However, the McLafferty technique does suffer from a number of disadvantages, include the following:
1) It is very difficult to confine positive ions and electrons simultaneously at the near thermal kinetic energies required for the ECD reaction to occur. This has until very recently only been accomplished in an ICR cell located within the high magnetic field of an FT-ICR mass spectrometer. These ECD ICR instruments use a superconducting magnet to generate magnetic fields typically on the order of 4.7 to 9 Tesla and therefore cost 0.5-1.5 million dollars each. Most protein sequence analyses are presently conducted on RF quadrupole ion trap, RF quadrupole linear trap, Q-TOF (quadrupole-time-of-flight), or TOF-TOF instruments. The primary difficultly with implementing ECD on any mass spectrometer other than an FTICR, is that the inhomogeneous RF field devices (RF traps and ion guides) conventionally used to contain ions during CAD will not confine electrons. This is because the mass of the electron is so small. Electrons injected into these devices also fail to remain at near thermal energies for a time interval that is sufficient to allow ECD reactions to occur with any efficiency. Accordingly, although some groups have recently reported performing ECD in RF ion traps, the sensitivity/fragment ion yield of these experiments is substantially lower than results obtained with conventional ECD.
2. ECD in the Fourier transform instruments is not very efficient. The best data we are aware of from the most advanced instruments indicates that the total (integrated) product ion signal is about 20% of that of the precursor (a precursor to product conversion efficiency of 20%). For comparison, commercial ion trap instruments, that utilize CAD, routinely produce precursor to product conversion efficiencies in the range of 50-100% depending on the precursor ion. Peptide ions generally have precursor to product conversion efficiencies on the higher end of this range. Instruments, such as the Q-TOF, that use RF-multipole, collision cell have somewhat lower precursor to product conversion efficiencies, but these are still generally in the range of 30-90%.
3. Most published ECD spectra are the averages (or sums) of several tens of recorded mass spectra. Typically a single FT/ICR spectrum takes on the order of a second to generate. This means that an ECD product-ion spectrum, having a reasonable signal to noise ratio, typically takes several tens of seconds to record. It probably takes at least 30 ions to create detectable ion signal. In contrast, RF quadrupole ion trap and Q-TOF type instruments, using electron multiplier-based detectors, readily detect single ions.
4. A further disadvantage of ECD is that when the precursor ions are large peptide or protein ions, the product ions of a given precursor often remain bound together, presumably by non-covalent bonds (hydrogen bonding) and fail to dissociate under ECD experimental conditions. A second activation (e.g., photo or collisional-activation) dissociation step is required to break these hydrogen bonds and to allow observation of ECD product ions (c and z-type product ions).
The present invention provides a new method of fragmenting positively charged peptides in an RF field mass spectrometer or an RF field ion containment device and for performing sequence analysis of peptides and proteins by mass spectrometry. The invention involves the use of a gas-phase anion to transfer an electron to a positively charged sample ion resulting in fragmentation of the positively charged sample ion.